Chelocardin derivatives

ABSTRACT

A number of specific hydrazone derivatives of chelocardin having high antibiotic activity are described. These new derivatives show unusually low toxicity and consequently, a very high therapeutic index as chemotherapeutic agents against gramnegative organisms.

United States Patent Inaba et al.

[111 3,907,889 Sept. 23, 19.75

CHELOCARDIN DERIVATIVES Inventors: Makoto Inaba; Edith Bernstein;

David Lyon Garmaise, all of Montreal, Canada Assignee: AbbottLaboratories, North Chicago, Ill.

Filed: June 18, 1973 Appl. N0.: 371,103

US. Cl...... 260/559; 260/453 AP; 260/50l.l4; 260/501.18; 260/50l.19;260/554; 260/562 H; 260/564 RF Int. Cl. C07C 103/19 Field ofSearch......... 260/559 AT, 554, 562 H, 260/564 RF References CitedFOREIGN PATENTS OR APPLICATIONS Germany Primary Examiner-C. DavisAttorney, Agent, or Firm-Paul D. Burgauer; Robert L. Niblack These newderivatives show unusually low toxicity and consequently, a very hightherapeutic index as chemotherapeutic agents against gram-negativeorganisms.

6 Claims, N0 Drawings l. CHELOCARDIN DERIVATIVES DETAILED DESCRIPTION OFTHE INVENTION Chelocardi'n is the name assigned to the antibiotic M-3l9originally described' in U.S. Pat. No. 3.155.582 issued in I964. Theoriginal publication did not disclose the chemical structure. but sincethen. the structure has been elucidated (see .I.A.C.S.. 92. page 6070 of1970) and as a result of this knowledge. new derivatives were prepared.Unfortunately, predicting physiological activity of such new derivativesis impossible but. surprisingly. anew group of compounds has been foundthat share and even exceed the chemotherapeutic activity of chelocardinitself while showing some advantageous physical and/or chemicalproperties.

The new compounds which are the subject of the present invention are theacyl and substituted aeylhy drazones. the guanylhydrazone. thesemicarbazone and to the base in routine and known fashion.

In order to illustrate the manner of preparing the new compounds.reference is made to the following examples which. however. are notintended to limit the invention in any respect. In all instances. thethin layer chromatography and spectrographic data obtained were inagreement with the assigned structures. Since the new substituent isattached in the 2a-position in all the derivatives described below. thislocation is not spethiosemicarbazone ofehelocardin. having the followingcifically indicated in these examples except by the general structure X(II) wherein R has the above meaning. in an inert, organic solvent oranaqueous mixture therewith. The term incrt" is used herein to expressthat ,the solvent does not reactor interfere with any of the startingmaterials or the formed end product. A preferred reaction medium isaqueous tetrahydrofuran (hereinafter simply referred to as THF);however, excellent results are also obtainedwhen the reaction medium ismethanol, ethanol or the like. .Usually. it is preferred to react asuitable salt ofchelocardin with the desired compound of formula II.c.g.. the desired hydrazide or hydrazide-related compound. 5 I

- In a preferred embodiment. a nontoxic acid addition salt ofchelocardin or chelocardin itself is combined with the compound offormula II in an inert, organic solvent and the solution is allowed tostand for at least minutes at room temperature. Reaction times beyond 24hours usually add no.further benefit and in many instances. thecondensation is essentially complete in l 2 hours. If desired. thetemperature. of the reaction medium may be raised but since roomtempershowing of its location in'formula l.

EXAMPLE I A solution of 0.83 g. of acethydrazide in 5 ml. of water isadded to 2.5 g. of chelocardin hydrochloride in I00 ml. of 98% aqueousTHF. The solution is decanted off a small quantity of solid impurity andallowed to stand for 1 hour at room temperature. The

4 condensation product crystallizes out of the solution in 50 andcooled. The crystalline chelocardin propionylhydrazone hydrochlorideseparates out in a yield of 2.04 g.; it melts at 235 238 C. withdecomposition.

EXAMPLE 3 Equimolar amounts of chelocardin hydrochloride andphenylacethydrazide are combined in tetrahydrofuran (175 ml. per mM).After standing for 1 hour, the solvent is evaporated and the residue isrecrystallized from ethanol. yielding 83% of chelocardinphenylacetylhydrazone hydrochloride. An analytically pure sample meltsat 222 225 C.

EXAMPLE 4 To a solution of 3 g. of chelocardin in [20 ml. of methanol isadded 1.07 g. of 4-aminobenzoylhydrazide. The mixture is stirred for 24hours at room temperature, filtered and the filtrate is concentrated to25 ml. The precipitated product is filtered, washed with ether and driedto produce a 5171 yield (2.0 g.) of chelocardin-4-aminobenzoylhydrazonehydrochloride.

EXAMPLE 5 A solution of 1.17 g. of 4-methoxybenzoylhydraiide in 60 ml.of methanol is added dropwise to a clear solution of 3 g. of chelocardinhydrochloride in 120 ml. ofmethanol. The mixture is then stirred for 15minutes.

filtered and the filtrate is concentrated to 5 ml. By adding 60 ml. ofwater. chelocardin 4-methoxybenzoylhydrazone hydrochloride precipitatesin a yield of 2.55 g. (64%).

EXAMPLE 6 The process of Example 5 is repeated with benzoylhydrazidereplacing the hydrazide used before. The solution is filtered through asmall bed ol'eharcoal before concentrating it. Yield: 2.1 g. (93%) ofchelocardin benzoylhydrazone hydrochloride: MP: 239 241 C. (dec.

EXAMPLE 7 Chelocardin hydrochloride (2.24 g.) is added in small portionsto a hot solution of 0.8 g. of 4-hydroxybenzoylhydrazide in 250 ml. ofmethanol containing 2 ml. of formic acid. After standing 30 minutes atroom temperature. the mixture is filtered through 2 g. of charcoal andthe filtrate is concentrated to 50 ml. in vacuo. By adding 100 ml. ofether. 2.64 g. of chelocardin 4-hydroxybcnzoylhydrazone hydrochloridemelting at 239 242 C. (dec.) is obtained.

EXAMPLE 8 ":A' solution of 0.8 g. of salicylhydrazide in 100 ml. ofmethanol is rapidly added to a stirred solution of 2.24 g. ofchelocardinhydrochloride in 120 ml. of methanol. The mixture is stirred 4 hours atroom temperature. brought to near boiling and filtered through 2 g. ofcharcoal. The filtrate is diluted with 100 ml. of ethanol and thesolution is concentrated to 100 ml. The precipitate formed is discarded;the solution is diluted with 300 ml. of ether which produces. uponstanding, 1.81 g. of pr'isms'identified as chelocardin2-hydroxybenzoylhydrazone hydrochloride melting at 237 241 C. (dec.).

EXAMPLE 9 A solution of 4. 1 2 g. of semicarbazide hydrochloride in40ml. ofwater is added to a stirred solution of 15.72 g. of chelocardinhydrochloride in 250 ml. of methanol. After standing for 2 hours, thesolution is filtered to yield 1 1.1 g. (60%) of chelocardinsemicarbazone hydrochloride.

EXAMPLE 10 To a solution ofO.89 g. of chelocardin hydrochloride in ml.of methanol is added 0.19 g. of thiosemicarbazide. The solution isrefluxed for 5 minutes, allowed to stand for 2 hours at room temperatureand diluted with ether to precipitate 0.92 g. (88 /1) of chelocardinthiosemicarbazone hydrochloride.

EXAMPLE ll By replacing the thiosemicarlmzide of Example 10 with 0.29 g.of nicotinylhydrazide. the procedure of Example 10 produces 815 yieldofchelocardin nicotinylhydrazone hydrochloride. melting at 234 236 C.(dec. 1. I g

EXAMPLE 12 A mixture of 0.895 g. of chelocardin hydrochlt-iride and 0.36g. of isonicotinylhydrazide in 5 ml. of water and 170 ml. of methanol isrefluxed for 4 hours. The insoluble material is filtered off and thefiltrate is concentrated to 15 ml. which produces 0.913 g. ofehelocardinisonicotinylhydrazone hydrochloride melting at 235 240 C. (dec.).

EXAMPLE 13 in a mixture of 25 ml. of formic acid and 25 ml. of THF. 1.64g. of chelocardin and 0.396 g. of eyanoacethydrazide are allowed tostand for 4 hours. By adding ether in portions of 50 ml.. ml. and ml..three fractions of precipitates are obtained. The middle fractionrepresents the desired chelocardin cyanoacethydrazone. Yield: 1.56 g.(7971); MP: 227 231 C. (dec.).

EXAMPLE 14 A solution of 631 mg. of furoylhydrazide in 10 ml. of

methanol is combinedwith 2.238 g of chelocardin hydrochloride in ml. ofmethanol. After stirring for 5 minutes. the mixture is allowed to standfor 45 minutes and is then concentrated to a viscous solution which isdiluted with ether. yielding-2.6 g. (93%) ofchelocardin2-furoylhydrazonehydrochloride; melting at 235 237 C. (dec:). I

EXAMPLELIES To a solution of l g. ofchelocardin in 40 ml. of methanol isadded 260 mg. zuninoguanidine hydrochloride After standing for 16 hoursat room temperature. the solution is filtered and the filtrateconcentrated to 5 ml. After diluting this concentrate with 35 ml. ofethanol. the volume is reduced to 25 ml. and the amorphous residue iscollected and combined with a second crop of material obtained afteraddition of ether. Yield: 940

mg. (78%) of chelocardin guanylhydrazone dihydro To'a suspension of1.777 g. of methylsemicarbazone and 2.0 g. of chelocardinhydrochloride-in 20.0 ml. of methanoL-lO ml. 0f2 N hydrochloride isadded and the mixture is heated under reflux for 27 hours and thencooled in an ice bath and filtered. The filtrate iscvaporated to about20 m1..in 4 steps and the solids produced are filtered after eachevaporation step. The firstcrop of 761 mg. and second crop of 785 mg.a'r'eiidenticalas established by lR-spectroscopic determination andmelting point. A total yield of1.456 g. (66.5%) ofchelocardin-methylsemicarbazone hydrochloride melting 5 at 238 240 C.(dec.) is obtained.

EXAMPLE 18 To a solution of 1.792 g. of chelocardin hydrochloride in 200ml. of methanol. a suspension of 2.243 g. of allylsemicarbazide in m1.of 2. N hydriochloric acid is added. The mixture is stirred at roomtemperature for 24 hours and subsequently filtered to remove theinsoluble solids. The filtrate is evaporated to dryness to yield 1.731g. i (79.4% of {chelocardinallylsemicarbazone hydrochloride melting at230 235 C. (dec.)

EXAMPLE 19 To a mixture of 1.788 g. of chelocardin hydrochloride and3.712 g. of o-ehl oroplre'nylsemiearbazi'de"in' 200 ml. of methanol,10ml. of'Z-N hydrochlorie'aeid is added and themixture is-stirred at'iooriftei'nperattirief' for 4 days. The mixture is thenlfil tereda'nd'the filtrate is evaporated stepwise-The first and seeond erop fl 7and 548 mg.. respectively. are identical as establish'e'd ZS by theirlR-spectra and melting point. A total yield of 1.265 g. (51.4%) ofchelocardin-o-chlorophenylsemicarbazone hydrochloride melting at 233 237C. (dec.) is obtained. I

EXAMPLE 20 z In order to show the antibiotic andbacteiziostatjc activity ofthe compounds of the-present 1I1YHI1OI13IP1 minimum inhibitoryconcentrations (MIC) z ire demonn strated in Table 1 below. The bacteriaarefirstgrown in a brain-heart infusion broth 01.24 hours .atthe.optimum temperature for the organism.'Thecaltureisthen diluted withwater so that therelare about-.1 0 :-Mio'. viable organisms perrni11iliter.. The cell suspcnsion isused: as the inoculum for the testsreported below. The test compounds. about 20 mg. of each. are dissolvedin 0.2 ml. of methanol and 19.8 ml. of water. The various test solutionsof varying concentrations are well distributed in agar suspensionsadjusted to a pH of 7.4 and placed in Petri dishes so that each dishcontains a known amount of test compound.

6 The surfaces ,of-the solidified agar plates are then inoculated withthe test culture by streaking thetest culture on the surface of theplate witha standardized loop that has been dipped inthe inoeulum andincubated at room temperature for 24 ho urs. The lYllC, values in Tablel are expressed in meg/ml. l I H As shown in Table 1. the compounds. ofthe present invention exhibit valuable bacteriostatie propertiesand areconsequentlyuseful in pharmaceutical compositions.- The eompoundsof.this inyention also cxhibi't very loworal and subcutaneous toxicitiesand produce essentially the same antibiotic activities in vivo aschelocardin.,Thus.;.the compounds-of. Examples 1. 13 and 15 showian oralC D CD mean curative dose) of below ..2 0(), n ig./kg. and,;:most .othercompounds shown above have CD values below 400-. mgjkg. as determined bythe mouse protection test. The same test shows subcutaneous CD values0125 rug/kg. while these values for the compounds of Examples 3. 9 and15 have a CD value of 12.5 25 mg./kg.; other compounds of this inventionshow subcutaneous CD values of around mg./kg.

1n additipn to the excellent .baeteriostatjic properties of thenefw'compounds. they also show a surprising physical characteristic:they. are more solublein water than chelocardinitself; some oft he ne'wco'mp'ounds. in fact. show extremely high watefsolubilities. In thisrespect. the new compounds distinguishfavorably over chelocardin itself.This excellent solubility makes the new compounds particularly suit-ablefor parenteral solutions which can easily be prepared by simplydissolving the new chelocardin derivatives in water which may bebuttered-to a pH 01,7.0 .7 8 and maycontain 0.5 5.71; by weight ofiaprese'rvative'such as benzyl a1 coholz. 5 s

Pre-ferably.' the new derivatives are usedin the fo rm of their :acidaddition salts with .pharmaceutical] acceptable acids. i.e..hydrochloric, sulfuric, acetic phosphoric. tartaric. citric. or;su;ceinjc-acid. since hydro- 0 chlorie acid forms stable acid addition saltswith the new compounds and such salts are suitable for pharmaceuticalpreparations and can be made easily. they are preferred For oral dosageforms, tablets, pills, wafers, suspensions, syrups. etc. can be preparedin standard fashion,

TABLE 1 Staph Staph. S. Pyo- Entero- Escher Kleb. Past. Prot. Prot.Salm. Compd. Aureus Aureus genes coccus Coli Pneum. Mult. Vulg. Mira.Typhi. of Ex. 45 Smith (2-203 89 Juhl 8045 10544 A88 .1] Fin. 9 Ed. 9

1 12.5 12.5v 6.2 12.5 12.5 6.2 0.78 6.2 6.2 6.2 2 50 50 25 50 50' 12.51.56 12.5 12.5 12.5 3 25 25 12.5 50 50 6.2 6.2 12.5 12.5 12.5 4 50 5O 2550 50 12.5 1.56 12.5 12.5 12.5 5 100 100 50 100 100 50 3.1 25 25 25 6100 100 25 100 100 25 3.1 25 25 25 7 50 50 50 100 50 25 3.1 12.5 12.5 258 100 100 100 100 100 100 25 100 100 100 9 25 25 25 50 25 12.5 1.56 12.525 6.2 10 50 50 25 100 100 25 3.1 50 50 25 11 100 100 100 100 100 10012.5 100 100 100 12 50 50 12.5 50 50 12.5 6.2 12.5 12.5 12.5 13 5O 50 5050 50 12.5 1.56 12.5 12.5 12.5 14 100 100 50 100 100 50 3.1 50 50 25 1550 50 25 50 25 "-12.5 3.1 12.5 12.5 25 16 25 25 12.5 50 25 6.2 0.78 6.26.2 6.2 17 5O 25 25 50 50 12.5 3.1 25 50 25 18 25 25 12.5 50 25 6.2 0.7812.5 6.2 12i6 19 100 50 25 100 100 50 6.2 50 50 50 using the usualpharmaceutically acceptable excipients such as carriers. diluents.pigments. dyes and coatings. The coatings for tablets may be of the kindthat dis solves rapidly in the acidic environment of the stomach. or asustained-release coating formulation may be selected to provide agradual release of the active ingredient over an extended period of timein order to maintain a bacteriostatic blood level over periods rangingfrom 2 24 hours.

For the treatment of smaller animals. a daily dose of It) 200 mg./kg. isrecommended for oral administration. For larger animals. includinghumans. a daily oral dose of St) 800 mg. produces the desired antibioticactivity. Oral dosages are preferably prepared in unit dosage form withthe dosage selected in such amounts that a single or several doses areadministered over a 24-hour period.

What is claimed is:

l. A compound of the formula:

with R cyanoloweralkyl.

loweralkyl. phenyl. benzyl. hydroxyphenyl. aminophenyl. or methoxyphenyl. and X represents =0. =5 or =NH, or a pharmaceuticallyacceptable acid addition salt thereof. 2. The compound of claim 1wherein R is methyl and X is O.

3. The compound of claim I wherein R is benzyl and representing X is O.

4. A compound as defined in claim I wherein X is oxygen.

5. The process of making a compound of the formula:

()H (l OH Y wherein R is loweralkyl. phenyl. hydroxyphenyl.methoxyphenyl. aminophenyl. or benzyl. cyanoloweralkyl. and X is O. S orNH or a nontoxic acid addition salt thereof. consisting essentially incombining chelocardin or a nontoxic acid addition salt thereof with acompound of the formula:

1. A COMPOUND OF THE FORMULA:
 2. The compound of claim 1 wherein R ismethyl and X is O.
 3. The compound of claim 1 wherein R is benzyl and Xis O.
 4. A compound as defined in claim 1 wherein X is oxygen.
 5. Theprocess of making a compound of the formula:
 6. The process of claim 5for making a compound of formula I wherein X is oxygen.